Sunday, October 6, 2019

Selenoproteins identified and quantified with TMT proteomics!

I sometimes think that my most positive attribute is that I have no problem accepting that I'm a moron. I got one sentence into this new-ish JASMS paper and was like -- "yup, I'm still totally dumb."

I quote: "The essential trace element selenium (Se) functions through it's incorporation as the 21st amino acid, selenocysteine (Sec; U), in proteins."

If I ever knew this fact, I think I should investigate the frequency of head impacts I suffer and maybe consider just wearing a helmet all the time.

There are a dozens of selenoproteins in humans alone and they are super important. Here is a nice 3 page open review on them (and a paragraph stolen from said review).

....head impacts.....

While I'm at it and since I have never once in my entire life considered looking for this -- yeah -- it's in UniMod.

This is the backdrop for this cool paper where these authors use TMT to study these proteins like this:

1) They raise mice on a purposely selenium deficient diet.
2) They take out the mouse macrophages  (I know how to do this and still have nightmares about doing it late at night in a creepy Hopkins sub-basement -- Aha! Proof I do remember something before 2013!)
3) They culture macrophages with and without Selenium
4) They activate the macrophages with LPS (the generic bacterial immune activator thing)
5) They extract the proteins, reduce, alkylate (with 2-chloroacetamide -- possibly important here) and TMT label as normal.

They use an Orbitrap Fusion with the TMT MS3 method under what looks like typical conditions. I don't want to read back through -- I should totally do something other than read about these today -- but I think they did 3 biological replicates.

1) They totally screwed up the abundance of the selenoproteins in their response to LPS activation!
2) My favorite part -- they never actually identify (and since it's TMT, thereby quantify) any peptides from the selenium region. (The image at the very top of this blog post shows -- no coverage of the 21st amino acid range!)

#1 is cool because -- holy cow -- if just depleting/supplementing an element can alter one of the most basic and critically important responses of the general immune system  --- what else does it do??!?

#2 I mean, obviously, right? If you're getting solid coverage of the rest of these proteins -- what on earth is going on at the Sec amino acid? How does the reduction, alkylation and labeling affect the Selenium? How well does a selenopeptide ionize? I'm sure one of the >27,000(!) references Google Scholar drags up for this has all these answers but still -- that's totally cool, right?

I'd probably be wasting a lot more time on my Sunday afternoon trying to figure this out, but the files aren't publicly uploaded.....

Okay -- I'm not sure why I'm so curious about this, but if you also are as well -- here is a great study where you can get the data files (found it through ProteomeXchange!).

The data for this study is Orbitrap Elite and all the files I downloaded have MS/MS in the ion trap so open searching and verification will take a lot longer than I have left free today. WHOA! The ETD MS/MS spectra are beautiful. This paper and corresponding dataset deserve some investigation.

Big question: HOW MANY MORE THINGS LIKE THIS (Selenocysteine!?!?) ARE THERE!?!??!  Hundreds? Thousands? Millions? More? All those unidentified spectra are seeming a lot less strange these days and might be bordering on surprising that unmodified peptide regions from any proteoforms exist at all....

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